This project studies mechanotransduction of platelets in health and diseases. Novel single-cell and single-molecule biophysical analyses will be employed to elucidate the mechanisms of mechanoreception by platelet glycoprotein (GP) Ib-IX-V complex. GPIb? mediates platelet rolling by binding to von Willebrand factor (VWF). Our preliminary studies showed that mechanical force exerted on GPIb?-VWF bonds can induce conformational changes in two regions of the GPIb? molecule. Importantly, these conformational change events were followed by induction of intraplatelet calcium fluxes. The duration, the magnitude, and the frequency of force were found to be important parameters in triggering platelet Ca2+ signaling. Furthermore, we identified an intermediate affinity state of integrin ?IIb?3 as induced by GPIb?-VWF interaction under force. We hypothesize that GPIb? functions as a mechanoreceptor such that force regulates its ligand binding and signaling processes. Dysregulation of GPIb? mechanoreception results in aberrant adhesion and signaling, leading to diseases in hemostasis and thrombosis. Previous methods of studying platelet signaling are of low spatial, temporal, and force resolutions, with insufficient capabilites for direct characterization of single- molecule dynamics, and unable to synchronize the adhesion and signaling observations at the single-platelet level. Our newly developed fluorescence biomembrane force probe technology overcomes these limitations. We will measure the kinetics of GPIb? interacting with VWF and simultaneously monitor signaling events including intracellular Ca2+ and cGMP levels as well as integrin activation. Three specific aims are proposed to test our hypothesis: 1) signal initiation by platelet mechanoreceptor GPIb?; 2) interplay between force-induced Ca2+ and cGMP signaling, and 3) dysregulated platelet mechanotransduction in human diseases. The proposed studies are innovative and unprecedented in several ways: in the ability to quantify signaling in single platelets in real-time concurrently with analysis of binding kinetics, and in defining the relationships among force attributes, bond characteristics, and signaling outcomes at the single-platelet and single-molecule levels. Importantly, we will elucidate the underlying mechanisms of the biophysical observation using mouse models targeting key molecular players of signaling pathways and relate dysregulation of mechanoreception by GPIb? with human diseases, including the bleeding disorder von Willebrand diseases (VWD) and thrombotic disorder thrombotic thrombocytopenic purpura (TTP). These studies will elucidate the force-regulated mechanreception of an important receptor on the platelet surface and thus provide key insights into vascular physiology and pathology, which will serve as a foundation for designing new therapeutic approaches to inhibiting pathological platelet function during thrombosis and/or intervention to VWD and TTP.